Compounds found to inhibit enzymes along metabolic pathways involving disease or pathways unique to pathogens may have useful bioactivity. Therefore, it is desirable to identify such inhibitors. Currently, assays of complete metabolic pathways in vitro are complicated by the kinetics of individual enzymes. Heretofore, component enzymes of a particular pathway were individually purified and assayed one by one. This individual assay approach also involved making the substrate for each enzyme separately. Further, attempts have been made to model behavior of metabolic pathways in vivo, in other words, by recreating the cellular environment. This approach, which has been used thus far only to study metabolic processes, has drawbacks as well. Specifically, one metabolic step is rate-limiting; therefore, when assaying using such a pathway model, it is more likely to find an inhibitor for one particular enzyme.
In particular, the present invention can be applied to the murein biosynthetic pathway. Compounds that inhibit enzymes along this pathway are expected to be antibiotics. Each gene in that pathway (murABCDEFGI, mraY, ddlA, alr) is essential for bacterial viability. The pathway is uniquely bacterial: no known eukaryotic homologues of these genes exist. There are known antibiotics (fosfomycin, cycloserine) whose molecular target is within the pathway. Additionally, this pathway is highly conserved amongst pathogenic bacteria, and thus it is expected that an inhibitor of this pathway will be a broad spectrum antibiotic.
Each of the genes in the murein biosynthetic pathway are known in the art. Table 1 lists each of the genes and their respective accession numbers in the GenBank/EMBL (European Molecular Biology Laboratory) database.
TABLE 1 ______________________________________ murA M92358, M76452 murB L14557 murC X52644 (also in X55034) murD X51584 (also in X55034) murE X55814 (also in X55034) murF X15432 (also in X55034) murG X52644 (also in X55034) murI L14556 mraY X55034 ddlA M58467 alr M12847 ______________________________________